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Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS

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Abstract

Reversed-phase ultrahigh-performance liquid chromatography-mass spectrometry (RP-UHPLC/MS) method was developed with the aim to unambiguously identify a large number of lipid species from multiple lipid classes in human plasma. The optimized RP-UHPLC/MS method employed the C18 column with sub-2-μm particles with the total run time of 25 min. The chromatographic resolution was investigated with 42 standards from 18 lipid classes. The UHPLC system was coupled to high-resolution quadrupole-time-of-flight (QTOF) mass analyzer using electrospray ionization (ESI) measuring full-scan and tandem mass spectra (MS/MS) in positive- and negative-ion modes with high mass accuracy. Our identification approach was based on m/z values measured with mass accuracy within 5 ppm tolerance in the full-scan mode, characteristic fragment ions in MS/MS, and regularity in chromatographic retention dependences for individual lipid species, which provides the highest level of confidence for reported identifications of lipid species including regioisomeric and other isobaric forms. The graphs of dependences of retention times on the carbon number or on the number of double bond(s) in fatty acyl chains were constructed to support the identification of lipid species in homologous lipid series. Our list of identified lipid species is also compared with previous publications investigating human blood samples by various MS-based approaches. In total, we have reported more than 500 lipid species representing 26 polar and nonpolar lipid classes detected in NIST Standard reference material 1950 human plasma.

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Abbreviations

BPI:

Base peak intensity

CAR:

Acylcarnitine

CE:

Cholesteryl ester

Cer:

Ceramide

CN:

Carbon number

DB:

Double bond

DG:

Diacylglycerol

DI:

Direct infusion

ECN:

Equivalent carbon number

ESI:

Electrospray ionization

FA:

Fatty acid

GlcCer:

Glucosylceramide

GM3:

Monosialodihexosylganglioside

HexCer:

Hexosylceramide

Hex2Cer:

Dihexosylceramide

Hex3Cer:

Trihexosylceramide

Hex4Cer:

Tetrahexosylceramide

HILIC:

Hydrophilic interaction liquid chromatography

HPLC:

High-performance liquid chromatography

Chol:

Cholesterol

IS:

Internal standard

LacCer:

Lactosylceramide

LPC:

Lysophosphatidylcholine

LPE:

Lysophosphatidylethanolamine

LPG:

Lysophosphatidylglycerol

LPI:

Lysophosphatidylinositol

LPS:

Lysophosphatidylserine

MG:

Monoacylglycerol

MS:

Mass spectrometry

MS/MS:

Tandem mass spectrometry

NP:

Normal-phase

-O:

Alkyl bond

-P:

Plasmalogen

PC:

Phosphatidylcholine

PE:

Phosphatidylethanolamine

PG:

Phosphatidylglycerol

PI:

Phosphatidylinositol

PS:

Phosphatidylserine

RIC:

Reconstructed ion current

RP:

Reversed-phase

SM:

Sphingomyelin

TG:

Triacylglycerol

tR :

Retention time

UHPLC:

Ultrahigh-performance liquid chromatography

UHPSFC:

Ultrahigh-performance supercritical fluid chromatography

QTOF:

Quadrupole-time-of-flight

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Funding

This work was supported by the grant project no. 21-20238S funded by the Czech Science Foundation.

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Authors and Affiliations

  1. Faculty of Chemical Technology, Department of Analytical Chemistry, University of Pardubice, Studentská 573, 53210, Pardubice, Czech Republic

    Zuzana Vaňková, Ondřej Peterka, Michaela Chocholoušková, Denise Wolrab, Robert Jirásko & Michal Holčapek

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  1. Zuzana Vaňková
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  2. Ondřej Peterka
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Correspondence to Michal Holčapek.

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Vaňková, Z., Peterka, O., Chocholoušková, M. et al. Retention dependences support highly confident identification of lipid species in human plasma by reversed-phase UHPLC/MS. Anal Bioanal Chem 414, 319–331 (2022). https://doi.org/10.1007/s00216-021-03492-4

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